Potravinarstvo Slovak Journal of Food Sciences

Potravinarstvo Slovak Journal of Food Sciences vol. 14, 2020, p. 721-728 https://doi.org/10.5219/1322 Received: 10 February 2020. Accepted: 22 April 2020. Available online: 28 September 2020 at www.potravinarstvo.com © 2020 Potravinarstvo Slovak Journal of Food Sciences, License: CC BY 3.0 ISSN 1337-0960 (online)

SURVEY OF MYCOBIOTA ON SLOVAKIAN GRAPES FROM SMALL CARPATHIANS WINE REGION

Soňa Felšöciová, Miroslava Kačániová, František Vrábel

ABSTRACT A total of 13 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2019 from Vrbové village in the Small Carpathian region of . For the isolation of fungi were used the direct plating technique on DRBC plates. The plates were incubated aerobically at 25 ±1 °C for one week in the dark. The data obtained from the cultivation of the grape berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the main components of the wine grape mycobiota of the Vrbovský subregion at harvest time (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis and Epicoccum (54%, each). The most abundant genera found by descending order were Penicillium (25%), Alternaria (24%), Cladosporium (20%), and Rhizopus (12%) and only in minor percentage by Aspergillus (3%) among others. The main fungal species isolated from genera Penicillium and Aspergillus were Penicillium expansum (57% RD) and A. section Nigri (97% RD). Of 17 analyzed Penicillium strains, 65% were able to produce at least one of the six mycotoxins analyzed in in vitro conditions by means of thin-layer chromatography method: citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C. Keywords: grape; filamentous fungi; Penicillium; mycotoxin; Slovakian

INTRODUCTION development in wine grapes brings significant yield losses is an important activity in many countries for , alters the chemical composition of wine (Einloft et al., 2017). Vine growing and viticulture have grapes, and produces secondary fungal metabolites and a very long tradition in Slovakia and are parts of the enzymes that together adversely affect wine flavor and country's cultural and historical heritage. Hundreds of color as well as yeast and lactic acid bacteria growth years of viticulture and viniculture have created a specific during vinification (Fleet, 2003). Among them, it is of type of landscape (Bezák et al., 2010), with unique great concern the presence of toxicogenic fungi in wine cultural and aesthetic values (Salašová and Štefunová, grapes capable of producing mycotoxins that could persist 2009). In total there exist six viticultural regions in during the winemaking process up to wine, being a high Slovakia with forty areas and wine-growing villages risk for consumer's health (Paterson et al., 2018; Prendes (ÚKSÚP, 2019a). Slovakia features almost 660 producers et al., 2015). growing around 13.500 ha of vines (of a potential The genus Alternaria is ubiquitously distributed and 15.300 ha) (ÚKSÚP, 2019b) for a production of about includes both saprophytic and opportunistic plant- 300.000 hL annually, which is primarily sold within the pathogenic species, which may affect crops in the field or national market. cause harvest and postharvest decay of plant products. The microflora of the grapes is highly variable, mostly Moreover, several Alternaria species are known to due to the influence of external factors as environmental produce toxic secondary metabolites, Alternaria parameters, geographical location, grape cultivars, and mycotoxins. The major Alternaria mycotoxins are the application of phytochemicals on the tetramic acid derivate, tenuazonic acid, and the (Pretorius, 2000; Pinto et al., 2014). A variety of fungal dibenzopyrone derivates, alternariol (AOH), and genera, mainly Botrytis, Alternaria, Aspergillus, alternariol monomethyl ether (AME) (Prendes et al., Penicillium, and Cladosporium, can contribute to grape 2015). Despite the toxic effects of the Alternaria toxins spoilage before harvest (Bellí et al., 2006; Magnoli et al., and their documented occurrence, they have not yet 2003; Medina et al., 2005). Filamentous fungi impact received the same attention as others mycotoxins and up to negatively in the production, sensory quality, and safety now, there is no regulation about them (EFSA, 2011). As characteristics of the wine in several ways. Their an opportunistic pathogen, it has the potential to cause a grape berry rot in the field under high disease pressure Volume 14 721 2020 Potravinarstvo Slovak Journal of Food Sciences situations. Strikingly, Alternaria has not been extensively colonies were identified to species level according to studied in wine grapes as a hazardous genus. micro and macroscopic criteria, using the keys of Klich Penicillium has gained attention as grapevine pathogens. (2002) and Pitt and Hocking (2009). Penicillium strains Penicillium expansum can cause rot in grapes, but does not were isolated and cultivated on MEA, CYA, Creatine- usually attack grapes before harvest. Aside from losses in Sucrose agar (CREA) (Samson et al., 2010) and Yeast fruit, this species is regarded as the major producer of Extract agar (YES) (Samson et al., 2010). The Penicillium patulin, although this species produces many other toxic colonies were identified to species level according to Pitt metabolites such as citrinin, roquefortine C or and Hocking (2009) and Samson and Frisvad (2004). chaetoglobosins among others (Andersen, Smedsgaard and Frisvad, 2004). Mycotoxin production Toxinogenity of selected isolates was screened in in vitro Scientific hypothesis conditions by means of thin-layer chromatography (TLC) Some of the fungal species occurring on grapes and grape according to Samson et al. (2002), modified by Labuda products can produce mycotoxins, so species identification and Tančinová (2006). Extracellular metabolites – citrinin is critical to predicting the potential mycotoxin griseofulvin and patulin were carried out on YES agar and contamination of grapes and wine. intracellular cyclopiazonic acid, penitrem A, and roquefortin C on CYA agar. At 14 days of incubation, five MATERIAL AND METHODOLOGY agar plugs (4 mm diameter) were cut from the edge of Study area a colony (extracellular metabolites) or cut from a colony Village Vrbové is located in the Vrbovský subregion in (intracellular metabolites) from each Petri plate and placed the Small Carpathian wine region. The Small Carpathian in an Eppendorf tube. The plags were extracted in 500 µL wine region is the most extensive of the six wine regions of chloroform-methanol (2:1, v/v) (Reachem, Slovak in Slovakia (vineyards are covering 4175 hectares) and is Republic). The content of the tubes was stirred for 5 min located in the southwestern part of Slovakia (ÚKSÚP, by Vortex Genie ® 2 (MO BIO Laboratories, Inc. – 2019b). Vines have been grown on the south-facing slopes Carlsbad, CA, USA). The extract of liquid phase 30 µL of the Small Carpathian mountains in locality Záhorie for along with 10 µL of standards (Sigma, Germany) was more than three thousand years. This region has a medium transferred to the TLC plate (Alugram ® SIL G, Macherey climate and abundant moisture. – Nagel, Germany). The plate was put into TEF solvent Last year, as a whole, was extremely warm. The year (toluene:ethyl acetate:formic acid – 5:4:1, toluene – 2019 had the same average annual temperature in Mikrochem, Slovak Republic; ethyl acetate and formic Hurbanovo 12.42 °C as in 2018. This value is a record acid – Slavus, Slovak Republic). After elution, the plate high for Hurbanovo since the record began. During the was air-dried. The identification of the metabolites was whole year, 2019 was only one month of the territory done by comparison with metabolite standards. temperature below normal. It was May (Beránek and Cyclopiazonic acid was visible directly in daylight after Faško, 2020). spraying with the Ehrlich reagent as a violet-tailed spot. Penitrem A was visible after spraying with 20% AlCl3 in 60% ethanol and heating at 130 °C for 8 min as a dark blue Grape sampling spot. Roquefortin C was visible after spraying with A total of 13 samples were taken: 3 from red varieties Ce(SO4)2 x 4 H2O as an orange spot. Patulin detection was (Alibernet, , and Blaufränkisch) and achieved by spraying with 0.5% methylbenzothiazolone 10 from white varieties (Palava, Green Veltliner, Seteasca hydrochloride (MBTH) (Merck, Germany) in methanol Regala, , Rheinriesling, , and heating at 130 °C for 8 min and then detected as Sauvignon, , Irsai Oliver, and Müller Thurgau). a yellow-orange spot under visible light. Citrinin was The sampling was conducted at the 2019 , at the detected directly as an intense yellow‐green streak under end of September. Two diagonals crossing the vineyards ultraviolet light (365 nm) as well as griseofulvin, which were delimited, and five healthy and undamaged bunches was visible as a blue spot. from each diagonal were obtained. Each bunch was collected in a sterilized plastic bag and sent to the laboratory chilled on ice. Statistical analysis The obtained results were evaluated and expressed according to relative density (RD) and isolation frequency Mycological analysis (Fr). The relative density (%) is defined as the percentage Fifty berries were selected randomly from each sample of isolates of the species or genus, occurring in the (totaling 650 berries) and placed in Dichloran Rose Bengal analyzed sample (Guatam, Sharma and Bhadauria, Chloramphenicol agar (DRBC) (Samson et al., 2002). 2009). These values were calculated according to Plates were incubated at 25 ±1 °C for 7 days in darkness. González et al. (1999) as follows: Genera identification was conducted according to RD (%) = (ni/Ni) x 100 microscopic and macroscopic criteria using the key of Pitt where ni – number of isolates of a species or genus; and Hocking (2009). Aspergillus strains were isolated and Ni – total number of isolated fungi. cultivated on MEA (Malt extract agar) (Samson et al., 2010), CYA (Czapek yeast extract agar) (Samson et al., The isolation frequency (%) is defined as the percentage 2010), and CY20S (Czapek yeast extract with 20% of samples within which the species or genus occurred at sucrose) (Pitt and Hocking, 2009). The Aspergillus

Volume 14 722 2020 Potravinarstvo Slovak Journal of Food Sciences least once. These values were calculated according to presence of P. expansum spores enhanced B. cinerea González et al. (1999) as follows: growth, while the latter avoided patulin accumulation. The data in Table 2 obtained from the cultivation of the Fr (%) = (ns/N) x 100; where ns – number of samples with berry samples revealed a high diversity of fungal species a species or genus; N – total number of samples. (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the most frequently occurring genera (92%, RESULTS AND DISCUSSION each), followed by Cladosporium (85%), Penicillium Fifteen fungal genera were identified from the grape (77%), Botrytis, and Epicoccum (54%, each). Penicillium samples: Alternaria, Aspergillus, Aureobasidium, Botrytis, spp. was predominant in terms of relative abundance Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, (25%), followed by Alternaria (24%), Cladosporium Phoma, Rhizopus, Syncephalastrum, Trichoderma, (20%), Rhizopus (12%), and Botrytis (6%). Besides, Trichothecium, and Ulocladium. About 2 % of the isolated a minor portion (<5%) of Aspergillus and other genera was fungi did not produce conidiophores or conidia on the found. tested conditions and were nominated as non-sporulated Alternaria genus was the main component of the wine fungi Mycelia sterilia. The number of isolates within the grape mycobiota of the Vrbovský subregion (Small several genera found on grapes from different varieties are Carpathian wine-growing region) at harvest time, which is shown in Table 1. The highest number of isolates (from in agreement with previous studies carried out in several 101 to 199) with 7, 8, or 9 genera were isolated from winemaking regions worldwide, e.g. from Uruguay varieties Müller Thurgau (13), Irsai Oliver (12), (Garmendia and Vero, 2016), Argentina (Magnoli et al., Blaufränkisch (3), Palava (4), and Alibernet (1). The lower 2003; Prendes et al., 2015), Spain (Medina et al., 2005), number of isolates (26, 28, respectively) were isolated Slovakia (Felšöciová et al., 2015c; Felšöciová, Mašková from the white variety Rheinriesling (8) with the number and Kačániová, 2018; Felšöciová and Kačániová, of genera 6 and Pinot Blanc (11) with the number of 2019a; Felšöciová and Kačániová, 2019b). genera 4. It is interesting to note the absence of isolates It was followed by Penicillium, which recorded a belonging to microscopic filamentous fungi in one sample frequency of 77% and a high relative density of 25%. Green Veltliner (5). This wine grape was colonized only From the previous study by Felšöciová and Kačániová by yeasts. All samples (except sample 5) were colonized (2019a), Penicillium contributed a small proportion (21% by genera Alternaria and Rhizopus. Genus Alternaria was Fr, <1% RD) from mycobiota associated with grapevine in dominated in samles Palava (4), Cabernet Sauvignon (2) Vrbové. The Botrytis genus, which is regarded as the main and Welschriesling (9), genus Rhizopus in sample spoilage cause in wine grapes, was isolated in this study, Alibernet (1), genus Penicillium in samples Müller but the absence of this genus has already been reported by Thurgau (13), Irsai Oliver (12), Blaufränkisch (3) and Magnoli et al. (2003) in Argentina, and Medina et al. Welschriesling (9) and genus Cladosporium in samples (2005) in Spain. Grey mold is responsible Irsai Oliver (12) and Sauvignon (10). for severe economic loss. Musts obtained from botrytized Thirty of the 32 Aspergillus isolates were identified as grapes are more liable to oxidation because of the A. section Nigri and 1 isolate as A. ochraceus. Sixteen polyphenol oxidizing activity of B. cinerea laccase and are black aspergilli were isolated from the Blaufränkisch not suitable for wine production (Morales et al., 2013). variety (3), 6 from Cabernet Sauvignon (2), 5 from Irsai Aspergillus was one the less common genera (46% Fr, Oliver (12), 2 from Palava (4), and 1 from Chardonnay (7). 3 % of all fungi). These results differ from those obtained Among section Nigri, A. carbonarius is considered the by other authors, who reported a much higher frequency predominant species responsible for the occurrence of from this genus, ranging from 70% to 95% (El Khoury et OTA in wine grapes and derivatives (Ponsone et al., al., 2008; Magnoli et al., 2003; Medina et al., 2005). 2010; Visconti et al., 2008). A low occurrence of this Data in Table 3 show that, 32 Aspergillus species were fungus was previously reported in Argentina (Chiotta et identified from grape samples. The section Nigri was al., 2009; Ponsone et al., 2010), Brazilia (Einloft et al., predominant within the Aspergillus genus, representing 2017) and Lebanon (El Khoury et al., 2006), and the 94% of species isolated from this genus with 38% absence of this fungus was observed in cold regions, like frequency. Certainly, the Aspergillus species are present Germany, North , Czech Republic and Portugal worldwide, in all the grape products and under all (Abrunhosa et al., 2001; Ostrý et al., 2007; Varga et al., environmental conditions (Somma, Perrone and 2005). Logrieco, 2012). From the 12 vineyards in the Small Three species of Penicillium were isolated from grapes. Carpathian area (14 samples), 79% of the samples were Species Penicillium expansum were dominated from the coloniezed by the genus Aspergillus (Felšöciová et al., Müller Thurgau variety (13), Blaufränkisch (3), Seteasca 2015c). During the 3 years survey (2011, 2012, and 2013), Regala (6), Rheinriesling (8), and Palava (4). Penicillium 37 isolates belonging to 7 Aspergillus species (A. clavatus, expansum has a high incidence in certain wine regions A. flavus, A. section Nigri, A. ostianus, A. parasiticus, such as bordering regions of North Portugal and Galiza A. versicolor and A. westerdijkiae) were isolated. The (Spain) (Serra et al., 2006). The incidence of P. expansum main occurring Aspergillus species of the samples were in some wine regions is high, but the attack of this fungus A. section Nigri (64%), as in our research. On the other to vineyards, is rare, being B. cinerea the most common hand, the most species were not been isolated from any of disease. Morales et al. (2013) observed that, in vitro, the the samples analyzed in the present study.

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Table 1 Fungi identified in Slovak wine grapes from exogenous mycobiota in 2019 by direct plating method. Fungal taxa Grape varieties 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Alternaria 17 28 36 77 - 13 10 7 25 7 2 4 26 Aspergillus - 7 16 2 - - 1 1 - - - 5 - A. ochraceus - 1 ------A. section Nigri - 6 16 2 - - 1 - - - - 5 - A. sp. ------1 - - - - - Aureobasidium ------1 Botrytis 17 8 - - - - 10 - - 7 6 4 12 Cladosporium 14 7 14 2 - 5 5 - 6 22 9 67 55 Epicoccum 5 3 1 - - 3 - - - 1 - 6 5 Fusarium 2 - 2 12 - - - - - 1 - - - Mucor - 4 - 3 - 1 4 2 - - - 3 - Penicillium 19 5 48 5 - 12 - 10 23 1 - 51 90 P. crustosum ------1 P. expansum - - 41 5 - 12 - 10 6 - - 15 61 P. griseofulvum ------10 - P. sp. 19 5 7 - - - - - 17 1 - 26 28 Phoma 1 - - - - - 1 - 1 - - - - Rhizopus 26 23 6 6 - 6 2 1 15 3 9 18 8 Syncephalastrum ------1 - - - Trichoderma - - 1 - - 2 5 - - - - 1 2 Trichothecium ------1 - - - - - Ulocladium - - - 2 - 1 ------Mycelia sterilia - - - - - 9 4 4 2 2 2 2 - Total isolates 101 85 124 109 - 52 42 26 72 45 28 161 199 Note: 1. Alibernet, 2. Cabernet Sauvignon, 3. Blaufränkisch, 4. Palava, 5. Green Veltliner, 6. Seteasca Regala, 7. Chardonnay, 8. Rheinriesling, 9. Welschriesling, 10. Sauvignon, 11. Pinot Blanc, 12. Irsai Oliver, 13. Müller Thurgau.

Table 2 The occurrence, isolation frequency and relative density of filamentous microscopic fungi in surface mycobiota of grapes (n = 13) harvested in Small Carpathian region. Fungal taxa No. Fr (%) RD (%) Alternaria 252 92 24 Aspergillus 32 46 3 Aureobasidium 1 8 <1 Botrytis 64 54 6 Cladosporium 206 85 20 Epicoccum 24 54 2 Fusarium 17 31 2 Mucor 17 46 2 Penicillium 264 77 25 Phoma 3 23 <1 Rhizopus 123 92 12 Syncephalastrum 1 8 <1 Trichoderma 11 38 1 Trichothecium 1 8 <1 Ulocladium 3 15 <1 Mycelia sterilia 25 54 2 Total isolates 1044 Note: No – number of isolated micromycetes, Fr – isolation frequency, RD – relative density.

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The toxicogenic profile of the 17 Penicillium isolates The occurrence, isolation frequency and Table 3 representing P. crustosum, P. expansum and relative density of Aspergillus species in surface P. griseofulvum from the Slovak grapes is shown in Table mycobiota of grapes (n = 13) harvested in Small 5. Carpathian region. The 65% of the 17 analyzed Penicillium strains were able Aspergillus species No. Fr (%) RD (%) to produce at least one of the six mycotoxins tested A. ochraceus 1 8 3 (citrinin, griseofulvin, patulin, cyclopiazonic acid, A. section Nigri 30 38 94 penitrem A, and roquefortin C). Citrinin was the toxin A. sp. 1 8 3 produced by the majority of the strains P. expansum Total isolates 32 (93%). It was followed by patulin produced by 79% of the Note: No. – number of isolates, Fr – isolation strains P. expansum, and roquefortin C produced by 64% frequency, RD – relative density. of the strains. Penicillium crustosum produced only penitrem A, did not produce roquefortin C. Two strains of Penicillium griseofulvum produced griseofulvin and Table 4 The occurrence, isolation frequency and patulin, the production of cyclopiazonic acid and relative density of Penicillium species in surface roquefortin C was confirmed by one isolate. mycobiota of grapes (n = 13) harvested in Small Almost 100% of Penicillium expansum strains are patulin Carpathian region. producers (Andersen, Smedsgaard and Frisvad, 2004; Penicillium species No. Fr (%) RD (%) Morales et al., 2008), which does not fully correspond to P. crustosum 1 8 <1 our results. Penicillium expansum is commonly associated P. expansum 150 54 57 with apple rot, production of geosmin − a well‐known P. griseofulvum 10 8 4 compound with a strong earthy smell, and patulin P. sp. 103 54 39 contamination in apple derivatives (Morales-Valle et al., Total isolates 264 2011). However, patulin has been reported in grapes (Moake, Padilla‐Zakour and Worobo, 2005), processed Note: No. – number of isolates, Fr – isolation grape juice (Scott, Fuleki and Harwig, 1977), and frequency, RD – relative density. fermenting wine (Majerus, Hain and Kölb, 2008;

Bragulat, Abarca and Cabañes, 2008), although the

occurrence in wine is low because it is well-known to be Table 5 Toxinogenity of selected Penicillium strains, degraded partially by the fermentation process (Moss and isolated from exogenous mycobiota of wine grapes. Long, 2002). Patulin mainly induces gastrointestinal Species C G P CA PA RC disorders including ulceration, distension, and bleeding. P. 1/1 0*/1** The compound provokes congestion and oedema of crustosum pulmonary, hepatic, and gastrointestinal blood vessels and P. 13/14 11/14 9/14 tissues. Subcutaneous injection of patulin produced local expansum sarcomas in rats and is classified in group 3 as not P. 2/2 2/2 1/2 1/2 classifiable as to its carcinogenicity to human by IARC griseofulvum (Varga et al., 2015). Note: * – number of isolates with ability to produce mycotoxin, ** - number of tested isolates, C – citrinin, CONCLUSION G – griseofulvin, P – patulin, CA – cyclopiazonic acid, Our results indicate a high diversity of fungal species PA – penitrem A, RC – roquefortin C. with a high incidence of Alternaria genus. Out of

17 potentially toxigenic Penicillium strains isolated from Data in Table 4 show 3 different Penicillium species exogenous mycobiota, namely P. crustosum, P. expansum from the 264 fungal strains. Penicillium expansum was and P. griseofulvum, 65% produced at least one mycotoxin predominant within the Penicillium species, representing by thin-layer chromatography method. The occurrence of 57% of the isolates and 54% frequency, which agrees with the potentially toxigenic fungus Aspergillus was overall previous publications by Felšöciová and Kačániová very low what indicates the high quality of the wine grapes (2019a), Felšöciová and Kačániová (2019b). The produced in Slovakia. predominant species of Penicillium isolated from grapes differs between vineyards. For example, Penicillium REFERENCES expansum is the species most frequently isolated in South Abrunhosa, L., Paterson, R. R. M., Kozakiewicz, Z., Lima, Slovak wine region (28% RD) (Felšöciová et al., 2017), P. N., Venâncio, A. 2001. Mycotoxin production from fungi aurantiogriseum in East Slovak wine region (34% RD) isolated from grapes. Letters in Applied Microbiology, vol. (Felšöciová et al., 2015a), P. chrysogenum in Small 32, no. 4, p. 240-242. https://doi.org/10.1046/j.1472- Carpathian wine region (64% RD) (Felšöciová et al., 765x.2001.00897.x 2015c), in Nitra wine region (28% RD) (Felšöciová et al., Andersen, B., Smedsgaard, J., Frisvad, J. C. 2004. 2013), in Central Slovak wine region (53% RD) Penicillium expansum: consistent production of patulin, (Felšöciová et al., 2014) and (39% RD) (Felšöciová chaetoglobosins, and other secondary metabolites in culture et al., 2015b). Penicillium expansum was found frequently and their natural occurrence in fruit products. Journal of in botrytized grapes (Morales-Valle et al., 2011). This Agricultural and Food Chemistry, vol. 52, no. 8, p. 2421- species was the second frequent (after P. chrysogenum) in 2428. https://doi.org/10.1021/jf035406k Tokaj (33% RD).

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Food Control, Contact address: vol. 57, p. 122-128. *Soňa Felšöciová, Slovak University of Agriculture, https://doi.org/10.1016/j.foodcont.2015.03.041 Faculty of Biotechnology and Food Sciences, Department Pretorius, I. S. 2000. Tailoring wine yeast for the new of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, millennium: novel approaches to the ancient art of Tel. +421376415813, winemaking. Yeast, vol. 16, no. 8, p. 675-729. E-mail: [email protected] https://doi.org/10.1002/1097- ORCID: https://orcid.org/0000-0002-2944-7071 0061(20000615)16:8<675::AID-YEA585>3.0.CO;2-B Salašová, A., Štefunová, D. 2009. Estetické atribúty vinohradníckej krajiny (Aesthetic attributes of the wine- Volume 14 727 2020 Potravinarstvo Slovak Journal of Food Sciences

Miroslava Kačániová, Slovak University of Agriculture František Vrábel, Slovak University of Agriculture, in Nitra, Faculty of Horticulture and Landscape Faculty of Biotechnology and Food Sciences, Department Engineering, Department of Fruit Sciences, Viticulture and of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, Enology, 2 Tr. A. Hlinku St., 949 76 Nitra, Slovakia, Tel. +421376415813, Department of Bioenergy and Food Technology, Faculty E-mail: [email protected] of Biology and Agriculture, University of Rzeszów, 4 ORCID: https://orcid.org/0000-0001-6780-2209 Zelwerowicza St., 35-601 Rzeszow, Poland, Tel. +421376414715, E-mail: [email protected] Corresponding author: * ORCID: https://orcid.org/0000-0002-4460-0222

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